Receptor Tyrosine Kinase FLT3 Research Articles

HOXA9 Regulome and Pharmacological Interventions in Leukemia.

HOXA9, an important transcription factor (TF) in hematopoiesis, is aberrantly expressed in numerous cases of both acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) and is a strong indicator of poor prognosis in patients. HOXA9 is a proto-oncogene which is both sufficient and necessary for leukemia transformation. HOXA9 expression in leukemia correlates with patient survival outcomes and response to therapy. Chromosomal transformations (such as NUP98-HOXA9), mutations, epigenetic dysregulation (e.g., MLL- MENIN -LEDGF complex or DOT1L/KMT4), transcription factors (such as USF1/USF2), and noncoding RNA (such as HOTTIP and HOTAIR) regulate HOXA9 mRNA and protein during leukemia. HOXA9 regulates survival, self-renewal, and progenitor cell cycle through several of its downstream target TFs including LMO2, antiapoptotic BCL2, SOX4, and receptor tyrosine kinase FLT3 and STAT5. This dynamic and multilayered HOXA9 regulome provides new therapeutic opportunities, including inhibitors targeting DOT1L/KMT4, MENIN, NPM1, and ENL proteins. Recent findings also suggest that HOXA9 maintains leukemia by actively repressing myeloid differentiation genes. This chapter summarizes the recent advances understanding biochemical mechanisms underlying HOXA9-mediated leukemogenesis, the clinical significance of its abnormal expression, and pharmacological approaches to treat HOXA9-driven leukemia.

Understanding the Role of Activation Loop Mutants in Drug Efficacy for FLT3-ITD.

The type III receptor tyrosine kinase FLT3 is a pivotal kinase for hematopoietic progenitor cell regulation, with significant implications in acute myeloid leukemia (AML) through mutations like internal tandem duplication (ITD). This study delves into the structural intricacies of FLT3, the roles of activation loop mutants, and their interaction with tyrosine kinase inhibitors. Coupled with this, the research leverages molecular contrastive learning and protein language modeling to examine interactions between small molecule inhibitors and FLT3 activation loop mutants. Utilizing the ConPLex platform, over 5.7 million unique FLT3 activation loop mutants-small molecule pairs were analyzed. The binding free energies of three inhibitors were assessed, and cellular apoptotic responses were evaluated under drug treatments. Notably, the introduction of the Xepto50 scoring system provides a nuanced metric for drug efficacy. The findings underscore the modulation of molecular interactions and cellular responses by Y842 mutations in FLT3-KD, highlighting the need for tailored therapeutic approaches in FLT3-ITD-related malignancies.

Allosteric SHP2 inhibition increases apoptotic dependency on BCL2 and synergizes with venetoclax in FLT3- and KIT-mutant AML

Mutations in the receptor tyrosine kinases (RTKs) FLT3 and KIT are frequent and associated with poor outcomes in acute myeloid leukemia (AML). Although selective FLT3 inhibitors (FLT3i) are clinically effective, remissions are short-lived due to secondary resistance characterized by acquired mutations constitutively activating the RAS/MAPK pathway. Hereby, we report the pre-clinical efficacy of co-targeting SHP2, a critical node in MAPK signaling, and BCL2 in RTK-driven AML. The allosteric SHP2 inhibitor RMC-4550 suppresses proliferation of AML cell lines with FLT3 and KIT mutations, including cell lines with acquired resistance to FLT3i. We demonstrate that pharmacologic SHP2 inhibition unveils an Achilles' heel of RTK-driven AML, increasing apoptotic dependency on BCL2 via MAPK-dependent mechanisms, including upregulation of BMF and downregulation of MCL1. Consequently, RMC-4550 and venetoclax are synergistically lethal in AML cell lines and in clinically relevant xenograft models. Our results provide mechanistic rationale and pre-clinical evidence for co-targeting SHP2 and BCL2 in RTK-driven AML.

Blockade of ROS production inhibits oncogenic signaling in acute myeloid leukemia and amplifies response to precision therapies.

Mutations in the type III receptor tyrosine kinase FLT3 are frequent in patients with acute myeloid leukemia (AML) and are associated with a poor prognosis. AML is characterized by the overproduction of reactive oxygen species (ROS), which can induce cysteine oxidation in redox-sensitive signaling proteins. Here, we sought to characterize the specific pathways affected by ROS in AML by assessing oncogenic signaling in primary AML samples. The oxidation or phosphorylation of signaling proteins that mediate growth and proliferation was increased in samples from patient subtypes with FLT3 mutations. These samples also showed increases in the oxidation of proteins in the ROS-producing Rac/NADPH oxidase-2 (NOX2) complex. Inhibition of NOX2 increased the apoptosis of FLT3-mutant AML cells in response to FLT3 inhibitors. NOX2 inhibition also reduced the phosphorylation and cysteine oxidation of FLT3 in patient-derived xenograft mouse models, suggesting that decreased oxidative stress reduces the oncogenic signaling of FLT3. In mice grafted with FLT3 mutant AML cells, treatment with a NOX2 inhibitor reduced the number of circulating cancer cells, and combining FLT3 and NOX2 inhibitors increased survival to a greater extent than either treatment alone. Together, these data raise the possibility that combining NOX2 and FLT3 inhibitors could improve the treatment of FLT3 mutant AML.

Synergistic Targeting of DNA-PK and KIT Signaling Pathways in KIT Mutant Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is the most common and aggressive form of acute leukemia, with a 5-year survival rate of just 24%. Over a third of all AML patients harbor activating mutations in kinases, such as the receptor tyrosine kinases FLT3 (receptor-type tyrosine-protein kinase FLT3) and KIT (mast/stem cell growth factor receptor kit). FLT3 and KIT mutations are associated with poor clinical outcomes and lower remission rates in response to standard-of-care chemotherapy. We have recently identified that the core kinase of the non-homologous end joining DNA repair pathway, DNA-PK (DNA-dependent protein kinase), is activated downstream of FLT3; and targeting DNA-PK sensitized FLT3-mutant AML cells to standard-of-care therapies. Herein, we investigated DNA-PK as a possible therapeutic vulnerability in KIT mutant AML, using isogenic FDC-P1 mouse myeloid progenitor cell lines transduced with oncogenic mutant KIT (V560G and D816V) or vector control. Targeted quantitative phosphoproteomic profiling identified phosphorylation of DNA-PK in the T2599/T2605/S2608/S2610 cluster in KIT mutant cells, indicative of DNA-PK activation. Accordingly, proliferation assays revealed that KIT mutant FDC-P1 cells were more sensitive to the DNA-PK inhibitors M3814 or NU7441, compared with empty vector controls. DNA-PK inhibition combined with inhibition of KIT signaling using the kinase inhibitors dasatinib or ibrutinib, or the protein phosphatase 2A activators FTY720 or AAL(S), led to synergistic cell death. Global phosphoproteomic analysis of KIT-D816V cells revealed that dasatinib and M3814 single-agent treatments inhibited extracellular signal–regulated kinase and AKT (RAC-alpha serine/threonine-protein kinase)/MTOR (serine/threonine-protein kinase mTOR) activity, with greater inhibition of both pathways when used in combination. Combined dasatinib and M3814 treatment also synergistically inhibited phosphorylation of the transcriptional regulators MYC and MYB. This study provides insight into the oncogenic pathways regulated by DNA-PK beyond its canonical role in DNA repair and demonstrates that DNA-PK is a promising therapeutic target for KIT mutant cancers.

Translatome Proteomics Identifies Autophagy As a Resistance Mechanism to on-Target FLT3 Inhibitors in Acute Myeloid Leukemia

Internal tandem duplications (ITD) in the receptor tyrosine kinase FLT3 are among the most frequent mutational targets in acute myeloid leukemia (AML), drive leukemia progression and confer a poor prognosis. Primary resistance to FLT3 kinase inhibitors (FLT3i) such as quizartinib, crenolanib and gilteritinib is a frequent clinical challenge and often occurs in the absence of identifiable genetic causes, suggesting that adaptive cellular mechanisms mediate primary resistance to on-target FLT3i therapy. We therefore systematically investigated acute cellular responses to on-target therapy with multiple FLT3i in FLT3-ITD+ AML using recently developed translatome proteomics to measure changes in the nascent proteome in combination with kinase signaling mapping by phosphoproteomics to identify therapy resistance mechanisms to FLT3 inhibitors in vitro. This systems-biology approach showed that the autophagy network escapes global translation repression caused by FLT3i and pinpointed autophagy induction mediated by the AKT-mTORC1-ULK1 axis as the most dominant stress response mechanism to on-target FLT3i therapy. FLT3i induced autophagy in a concentration- and time-dependent manner specifically in FLT3-ITD+ cells in vitro and in primary human AML cells ex vivo. Inhibition of FLT3-ITD signaling led to decreased phosphorylation at AKT S473, decreased phosphorylation at the AKT substrate mTOR S2448, and decreased phosphorylation at the mTORC1 substrate ULK1 S757. CRISPR/Cas9-targeting of TSC2, a negative mTORC1 regulator, ULK1 or ATG3, a core autophagy protein required for autophagosome formation, significantly attenuated or largely abolished autophagy induction by FLT3i. Pharmacological autophagy inhibition (ROC-325, Lys05) or genetic ablation of autophagy (shRNA-mediated knockdown of ATG3, ULK1 or its target Beclin1) increased the sensitivity to FLT3-targeted therapy in cell lines, patient-derived xenografts and primary AML cells ex vivo. In immunodeficient mice xenografted with human FLT3-ITD+ AML cells, oral co-treatment with gilteritinib and autophagy inhibitor ROC-325 synergistically impaired leukemia progression and extended overall survival. Our findings show the utility of functional translatome proteomics to systematically investigate cell-autonomous therapy resistance mechanisms, identify a molecular mechanism responsible for primary on-target FLT3i treatment resistance and demonstrate the pre-clinical efficacy of a rational combination treatment strategy targeting both FLT3 and autophagy induction.

Targeting of SFK and G6PD Contributes to Anti-Leukemic Activity of Novel FLT3 Trisubstituted Purine Inhibitors: Effective Therapeutic Approach for AML with FLT3 Mutations

We have recently developed compound LGR3922, a potent inhibitor of FLT3 receptor tyrosine kinase, which displays selectivity and potency in in vitro and in vivo AML models (compound 7d, Gucky T et al, J Med Chem. 2018;61(9):3855). In addition, LGR3922 showed strong inhibitory activity against Src family kinases (SFK) in the in vitro settings (Fig. 1A; ASH meeting abstract, Radosova Muchova et al, Blood. 2016;128(22):1569). Here, we present additional characterization of the lead compound LGR3922 and demonstrate its potent antileukemic activity in MLL/FLT3-ITD cell lines, murine xenografts and ex vivo primary AML samples. LGR3922 exhibited strong anti-proliferative effect against MV4-11 and MOLM-13 cells. Contrary to quizartinib (quiz), LGR3922 simultaneously reduced phosphorylation levels of FLT3-SFK and their downstream molecules STAT5 and ERK in both AML cell lines. Interestingly, the inhibitory effect of LGR3922 on SFK, STAT5 and ERK increased over time, especially in the MOLM-13 cells. Similar results were only seen for the combination treatment with quiz and dasatinib (das). Dual-targeting of FLT3-SFK by LGR3922 was further confirmed on MV4-11 xenografts. In the long-term in vivo experiment with repeated doses of the tested inhibitors, LGR3922 showed similar trends in initial treatment response as the combination treatment with quiz and das; quiz exhibited less efficient inhibitory effects at this stage. The tumor reduction was most significant (p ≤ 0.0001) in LGR3922 group; 3 out of 6 tumors were completely eradicated during the treatment period (Fig. 1B). Complete tumor eradication (in 1 out of 5 animals) over the treatment period was then seen only in combination quiz and das (Fig. 1B). No adverse effects on blood count parameters or body weight were associated with LGR3922 compared to combination treatment with quiz and das, where temporary body weight loss and only limited weight gain were observed. Next, we searched for additional target of the FLT3/SFK inhibitor that would have a synthetic lethal effect for the kinase pair. Besides the kinases that belong to other druggable nodes of AML-associated networks (Fig. 1A), we also tested antioxidant defense enzymes, previously validated as being synthetic lethal with FLT3 inhibitor therapy (Gregory MA et al. Proc Natl Acad Sci U S A. 2016;113(43):E6669). Indeed, we observed that LGR3922 inhibited G6PD activity, already after one-hour exposure (with a dose response over the concentration range), in contrast to quiz and das. We hypothesize that this activity modulating leukemia cell redox metabolism, cooperating with FLT3/SFK inhibition, may elicit more effective eradication of AML cells than quiz and das or quiz/das combination. We then evaluated toxicity of LGR3922 on normal hematopoiesis using colony forming unit (CFU) assays with progenitor cells from several healthy donor controls. LGR3922 inhibited colony formation in a dose-dependent manner; maximal tolerated concentration (MTC) for all healthy samples was 100 nM, in some samples the reduced granulocyte-macrophage (CFU-GM) and burst forming unit-erythroid (BFU-E) colony forming ability was not observed up to a concentration of 200 nM. Quiz and das exposure showed MTCs at 20 nM and 5 nM, respectively; in quiz/das combination at 10 nM/0.5 nM; 10 nM/5 nM was already toxic. Then, we performed CFU assays on 4 primary human AML samples with FLT3-ITD mutations (n=3) or FLT3-TKD+KMT2A::MLLT3 (n=1). Cultures with LGR3922 at 100 nM reduced the number of colonies by ∼70% in all FLT3-ITD patient samples. The inhibitory effects were comparable to or a little more potent than MTCs of quiz or quiz/das combination. Das culture had significantly lower effect on colony formation for the tested concentrations. In the FLT3-TKD+KMT2A::MLLT3 positive sample, fusion transcript was quantified in harvested colonies. While LGR3922 significantly reduced KMT2A::MLLT3 fusion transcript, no effects were observed for MTCs of single agent cultures with quiz or das. Only a combination of quiz/das exceeding MTC (10 nM/5 nM) resulted in a slight decrease of KMT2A::MLLT3 fusion transcript levels. Taken together, these data suggest effectiveness of our new LGR3922 compound and provide a rationale for further development and testing of this molecule for MLL/FLT3-ITD AMLs, and, potentially, other types of AMLs. Grant support: Program EXCELES, ID Project No. LX22NPO5102; GA19-09086S; MH CZ-DRO (UHKT, 00023736). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Disrupting PTPRJ transmembrane-mediated oligomerization counteracts oncogenic receptor tyrosine kinase FLT3 ITD.

The receptor protein tyrosine phosphatase (RPTP) PTPRJ (also known as DEP-1) has been identified as a negative regulator of the receptor tyrosine kinase FLT3 signalling in vitro. The inactivation of the PTPRJ gene in mice expressing the constitutively active, oncogenic receptor tyrosine kinase FLT3 ITD aggravated known features of leukaemogenesis, revealing PTPRJ's antagonistic role. FLT3 ITD mutations resulting in constitutively kinase activity and cell transformation frequently occur in patients with acute myeloid leukaemia (AML). Thus, in situ activation of PTPRJ could be used to abrogate oncogenic FLT3 signalling. The activity of PTPRJ is suppressed by homodimerization, which is mediated by transmembrane domain (TMD) interactions. Specific Glycine-to-Leucine mutations in the TMD disrupt oligomerization and inhibit the Epidermal Growth Factor Receptor (EGFR) and EGFR-driven cancer cell phenotypes. To study the effects of PTPRJ TMD mutant proteins on FLT3 ITD activity in cell lines, endogenous PTPRJ was inactivated and replaced by stable expression of PTPRJ TMD mutants. Autophosphorylation of wild-type and ITD-mutated FLT3 was diminished in AML cell lines expressing the PTPRJ TMD mutants compared to wild-type-expressing cells. This was accompanied by reduced FLT3-mediated global protein tyrosine phosphorylation and downstream signalling. Further, PTPRJ TMD mutant proteins impaired the proliferation and in vitro transformation of leukemic cells. Although PTPRJ's TMD mutant proteins showed impaired self-association, the specific phosphatase activity of immunoprecipitated proteins remained unchanged. In conclusion, this study demonstrates that the destabilization of PTPRJ TMD-mediated self-association increases the activity of PTPRJ in situ and impairs FLT3 activity and FLT3-driven cell phenotypes of AML cells. Thus, disrupting the oligomerization of PTPRJ in situ could prove a valuable therapeutic strategy to restrict oncogenic FLT3 activity in leukemic cells.

Activating Mutation in the Receptor Tyrosine Kinase FLT3 with Clinicopathological Relevance in Canine Mast Cell Tumors.

Recent research has focused on the receptor tyrosine kinase (RTK) KIT which is involved in the pathogenesis of canine mast cell tumors (MCT). However, the role of other RTKs in this neoplasm remains unclear. The present study aimed to determine the frequency of FLT3 mutations and to evaluate the mutational status and clinicopathological relevance of canine MCT patients. There were a total of 20 cases that were cytologically and histopathological diagnosed as canine MCTs; genomic polymerase chain reaction (PCR) and Sanger sequencing were used to identify mutations. For the juxtamembrane (JM) domain, the FLT3 14/15 primer pair was used to investigate exon 14/15 loci. Based on genomic PCR amplification of exon 14/15 and 20 of the FLT3 gene and Sanger sequencing of 20 cases of canine MCTs, the overall frequency of FLT3 mutation in canine MCTs was 75%. The majority of FLT3 mutations (70%) were internal tandem duplications (ITD) of the JM domain, while one case arose from deletion mutations of the tyrosine kinase domain (TKD). However, double mutations were not observed in this study. Furthermore, there is also clinicopathological relevance to MCT dogs carrying FLT3-ITD mutations, showing a tendency toward leukocytosis due to neutrophilia, and resembling human acute myeloid leukemia (AML) with FLT3-ITD mutations. A subset of MCTs with FLT3-ITD mutations, showing an enhanced signal of phosphorylated ERK1/2 identified by immunoblotting, suggests that an activating mutation may be driven by a distinct signal of the ERK pathway. Our results indicate that FLT3-ITD mutation is an oncogenic driver of canine MCTs, and that it shares some clinicopathologic features with human AML. These findings may offer new opportunities for further studies on canine mast cell tumorigenesis and a novel therapeutic target for canine MCT cases harboring FLT3-ITD mutations.

Translatome proteomics identifies autophagy as a resistance mechanism to on-target FLT3 inhibitors in acute myeloid leukemia

Internal tandem duplications (ITD) in the receptor tyrosine kinase FLT3 occur in 25 % of acute myeloid leukemia (AML) patients, drive leukemia progression and confer a poor prognosis. Primary resistance to FLT3 kinase inhibitors (FLT3i) quizartinib, crenolanib and gilteritinib is a frequent clinical challenge and occurs in the absence of identifiable genetic causes. This suggests that adaptive cellular mechanisms mediate primary resistance to on-target FLT3i therapy. Here, we systematically investigated acute cellular responses to on-target therapy with multiple FLT3i in FLT3-ITD + AML using recently developed functional translatome proteomics (measuring changes in the nascent proteome) with phosphoproteomics. This pinpointed AKT-mTORC1-ULK1-dependent autophagy as a dominant resistance mechanism to on-target FLT3i therapy. FLT3i induced autophagy in a concentration- and time-dependent manner specifically in FLT3-ITD + cells in vitro and in primary human AML cells ex vivo. Pharmacological or genetic inhibition of autophagy increased the sensitivity to FLT3-targeted therapy in cell lines, patient-derived xenografts and primary AML cells ex vivo. In mice xenografted with FLT3-ITD + AML cells, co-treatment with oral FLT3 and autophagy inhibitors synergistically impaired leukemia progression and extended overall survival. Our findings identify a molecular mechanism responsible for primary FLT3i treatment resistance and demonstrate the pre-clinical efficacy of a rational combination treatment strategy targeting both FLT3 and autophagy induction.

Flt3 Signaling in B Lymphocyte Development and Humoral Immunity

The Class III receptor tyrosine kinase Flt3 and its ligand, the Flt3-ligand (FL), play an integral role in regulating the proliferation, differentiation, and survival of multipotent hematopoietic and lymphoid progenitors from which B cell precursors derive in bone marrow (BM). More recently, essential roles for Flt3 signaling in the regulation of peripheral B cell development and affinity maturation have come to light. Experimental findings derived from a multitude of mouse models have reinforced the importance of molecular and cellular regulation of Flt3 and FL in lymphohematopoiesis and adaptive immunity. Here, we provide a comprehensive review of the current state of the knowledge regarding molecular and cellular regulation of Flt3/FL and the roles of Flt3 signaling in hematopoietic stem cell (HSC) activation, lymphoid development, BM B lymphopoiesis, and peripheral B cell development. Cumulatively, the literature has reinforced the importance of Flt3 signaling in B cell development and function. However, it has also identified gaps in the knowledge regarding Flt3-dependent developmental-stage specific gene regulatory circuits essential for steady-state B lymphopoiesis that will be the focus of future studies.

Abstract 3969: Y842 mutations in the activation loop of FLT3 regulates drug response in FLT3-ITD positive acute myeloid leukemia

Abstract Acute myeloid leukemia (AML) is an aggressive form of blood cancer with a poor prognosis. Approximately 30% of AML patients carry mutations in the type III receptor tyrosine kinase FLT3. The most common form of FLT3 mutations includes internal tandem duplication (ITD) mutations, which are also associated with poor clinical outcomes. Several inhibitors of FLT3 have been developed and are under clinical trials. Major problems in targeting FLT3-ITD include acquired resistance to the inhibitors which is mediated by secondary point mutations in FLT3. The activity of many, but not all, tyrosine kinases is dependent on the well-conserved tyrosine residue in the activation loop for receptor activation. For instance, we have recently shown that the KIT receptor is dependent on its activation loop tyrosine for downstream signaling and receptor stability, but not for the activation of the receptor. In many AML patients, FLT3-ITD mutations in combination with mutations in the activation loop tyrosine residue (Y842) result in drug resistance. We therefore stably transduced cells with FLT3-ITD in combination with different Y842 mutants (Y842C and Y842F). Using MM-PBSA, we found that the introduction of the Y842C and Y842F point mutations changed the binding energy of the FLT3 inhibitors quizartinib, sorafenib, and midostaurin in silico. In addition, the mutants displayed a change in drug response and apoptosis levels after treatments with FLT3 inhibitors in vitro. Previous data from our lab also shows that the Y842F mutant resulted in delayed tumor formation in vivo, indicating that the phosphorylation of the activation loop tyrosine is an important event in FLT3-ITD-mediated transformation. All in all, our data suggest that the activation loop tyrosine residue in FLT3-ITD positive cells plays an important role in drug response and tumor formation. Citation Format: Lina Al Ashiri, Sausan Moharram, Rituraj Purohit, Julhash Kazi Uddin, Lars Rönnstrand. Y842 mutations in the activation loop of FLT3 regulates drug response in FLT3-ITD positive acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3969.

Efficacy of midostaurin combined with intensive chemotherapy followed by allogeneic hematopoietic stem cell transplantation in a patient with NPM1 and FLT3-TKD mutated acute myeloid leukaemia with clinical high-risk features

Many genetic disorders occur in patients suffering from acute myeloid leukaemia (AML). The most common mutations found in such patients are in the nucleophosmin ( NPM ) gene and the FLT3 tyrosine kinase receptor gene. NPM1 mutation is observed in 30–35% of adult AML patients (50–60% of AML with normal karyotype), showing a favourable prognosis. The presence of point FLT3 comutations occur in the tyrosine kinase domain (TKD) are associated with even more favourable prognosis. However FLT3 comutations i.e. internal tandem duplication (ITD) in particular with high allelic ratio (ITD high ) worsen the course of leukemia and treatment outcomes. Deploying FLT3 tyrosine kinase inhibitors thus offers a prospect for improving treatment and prolonging the survival of patients with AML, burdened with the FLT3 gene mutation. Midostaurin and gilteritinib are type I FLT3 inhibitors which are used to treat patients with AML FLT3 -TKD due to their mechanism of action. This paper presents the case of a 30-year-old AML patient diagnosed with NPM1 and FLT3 -TKD mutations, bone marrow reticuline fibrosis and extramedullary sites of AML. Treatment was individualized and induction chemotherapy was combined with midostaurin. After first-line treatment with midostaurin, complete remission was achieved, as confirmed by histopathological examination of the bone marrow. Subsequently, two cycles of consolidation chemotherapy were given, and allogeneic haematopoietic stem cells were transplanted from an unrelated donor after myeloablative conditioning. The patient has remained in complete leukaemia remission, 3 years after diagnosis.

Identification of the Benzoimidazole Compound as a Selective FLT3 Inhibitor by Cell-Based High-Throughput Screening of a Diversity Library.

Internal tandem duplication in the FLT3 receptor tyrosine kinase (FLT3/ITD mutation) occurs in approximately 25% of acute myeloid leukemia (AML) patients. To specifically target this driver mutation in AML, we assessed and compared the cell-based cytotoxicity of a diversity library (10,000 compounds) against the normal cell line BaF3 and the isogenic leukemic cell line BaF3/ITD. A benzoimidazole scaffold-based compound (HP1142) was identified as the most selective compound against a series of murine and human leukemia cells with FLT3/ITD. Novel benzoimidazole compounds were further designed to improve the aqueous solubility of HP1142. The most potent compound, HP1328, demonstrated desirable pharmaceutical and pharmacokinetic properties. Treatment with HP1328 significantly reduced the leukemia burden and prolonged the survival of mice with FLT3/ITD leukemia. Our findings establish the specific activity of the benzoimidazole compound against FLT3/ITD leukemia and warrant further investigation in this subset of leukemia patients with poor prognosis.

Real-World Outcomes with Fedratinib Therapy in Patients Who Discontinued Ruxolitinib for Primary Myelofibrosis

▪Introduction: Myelofibrosis (MF) is a rare myeloproliferative disorder in whichdysregulation of the janus kinase 2 (JAK2) hematopoiesis-signaling pathway disruptsbone marrow production of blood cells, leading to anemia, fatigue and splenomegaly.Until recently, the dual JAK1/JAK2 inhibitor, ruxolitinib (RUX), has been the onlyapproved pharmacological therapy for intermediate/high-risk MF. However, manypatients receiving RUX experience a suboptimal response or develop cytopenia, leadingto discontinuation. Fedratinib (FEDR), an oral, selective inhibitor with activity againstwild-type and mutationally activated JAK2 and the receptor tyrosine kinase FLT3, wasapproved in the US in August 2019. The real-world effectiveness of FEDR in the post-RUX setting has not yet been assessed.Aim: To assess the baseline characteristics and survival outcomes among patientsdiagnosed with MF who discontinued RUX and were subsequently treated with FEDR ornot.Methods: Adult US patients who received RUX after an initial diagnosis of primary MF(identified by ICD-9/10-CM codes) were identified using Flatiron Health's nationwideelectronic health record (EHR)-derived database. Patients were stratified by whetherthey subsequently received post-ruxolitinib fedratinib (FEDR group) or not (non-FEDRgroup). The non-FEDR group was further stratified by time of RUX discontinuation(before [non-FEDR subgroup A] and after [non-FEDR subgroup B] US approval of FEDRin August 2019) to enable a contemporaneous comparison with the FEDR group. Indexdate was the start of FEDR therapy in the FEDR group, and last date of RUX therapy inthe non-FEDR groups. Patients were included if they had ≥2 recorded visits in theFlatiron network on or after January 1, 2011- Oct 31, 2020; were aged ≥18 years atindex; had ≥1 months' data before and also after index; and had no record of receivingunclassified clinical study drugs prior to index. Patient demographics and clinical characteristics were assessed at index. Overall survival (OS) was defined as time fromindex until death or censoring, and was assessed by Kaplan-Meier analysis. Landmarksurvival was defined as the proportion of patients who survived at a given point.Association of baseline variables and survival were assessed by Cox proportionalhazards model and p<0.05 was considered statistically significant.Results: Overall, 229 MF patients were evaluated (FEDR group: n=70; non-FEDRgroup: n=159). The median age at index for the FEDR group and non-FEDR group was71.0 and 70.0 years, 55.7% and 50.3% were female, 64.3% and 68.6% were white, andmedian follow-up from index was 7.0 and 6.0 months, respectively. Among patients withEastern Cooperative Oncology Group performance status (ECOG PS) at index, 90.2%(46/51) and 74.3% (84/113) had an ECOG score of 0-1, respectively (Table). Median(range) duration of FEDR therapy in the FEDR group was 3.7 (0-12.2) months; 47.1%(33/70) of patients receiving FEDR initiated therapy with 400 mg FEDR once daily, and21.4% (15/70) initiated with 200 mg FEDR once daily. Median OS was not reached in theFEDR group and was 17 months in the non-FEDR group. Landmark survival in theFEDR and non-FEDR groups was 91.3% and 76.5% at 3 months, and 71.6% and 53.5%at 12 months, respectively. In the non-FEDR subgroup B, the corresponding survivalrates at 3 months and 12 months were 75.0% and 47.9%. The Cox proportional hazardsmodel suggested that being male, ‘other’ (non-white, non-black/African-American, non-Asian, non-missing) race, Charlson comorbidity index ≥1, ECOG score 2-4, and bodymass index <18.5 kg/m were significantly associated with poorer survival.Conclusions: This real-world analysis demonstrates that these two groups of patientswith primary MF who had received prior RUX had broadly comparable baselinecharacteristics. FEDR appeared to be associated with improved survival up to 1 yearafter index compared with non-FEDR therapy. When the FEDR group and non-FEDR Bsubgroup were compared, the differences in survival rate remained. FEDR may offersubstantially improved survival probability in patients receiving post-ruxolitinib therapy of primary MF in routine clinical practice. [Display omitted] DisclosuresPassamonti: Novartis: Research Funding, Speakers Bureau; Celgene: Research Funding, Speakers Bureau. Do: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Lou: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Chevli: Bristol-Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Abraham: Bristol Myers Squibb: Current Employment.

Allosteric SHP2 Inhibitor RMC4550 Synergizes with Venetoclax in FLT3 and KIT Mutant Acute Myeloid Leukemia

Mutations in receptor tyrosine kinases (RTK) FLT3 and KIT occur frequently in Acute Myeloid Leukemia (AML) and are associated with high risk of relapse. FLT3 tyrosine kinase inhibitors (TKI) are clinically approved in AML, but resistance is common and involves emerging clones reliant on oncogenic signaling, particularly in the RAS/MAPK pathway. Patients who relapse on FLT3 TKIs have inauspicious prognoses and no specific therapeutic options, highlighting the unmet need for effective strategies to target oncogenic signaling and improve outcomes in relapsed/refractory (R/R) AML.The protein tyrosine phosphatase SHP2 (PTPN11) is a central node in RAS/MAPK activation downstream of various RTKs, including FLT3, acting as a scaffold for adaptor proteins that promote RAS-GTP loading. Novel allosteric inhibitors are being clinically investigated in cancers with signaling activating mutations.Here, we demonstrate that the allosteric SHP2 inhibitor RMC-4550 modulates expression of pro and anti-apoptotics in FLT3 and KIT mutant AML providing rationale for combinatorial targeting of SHP2 and BCL2 as a synergistic approach. We subsequently report the preclinical efficacy of RMC-4550 and the FDA-approved, BCL2 selective inhibitor, Venetoclax combination in both in vitro and in vivo AML models.We evaluated cell viability of multiple AML cell lines treated with RMC-4550. FLT3-ITD (Molm14, MV4-11) and KIT mutant (Kasumi1, SKNO1) lines were sensitive to SHP2 inhibition. RMC-4550 maintained its efficacy in FLT3-ITD Molm14 cells with secondary mutations in FLT3 tyrosine kinase domain (TKD) and in NRAS G12C. RMC-4550 biochemically represses pERK (Figure 1A) and transcriptionally downregulates mRNA expression of DUSP6 and anti-apoptotic BCL2 and MCL1.We functionally evaluated the mitochondrial outer membrane permeabilization (MOMP) in response to SHP2 inhibition using a dynamic iBH3 profiling assay. RMC-4550 increased the overall priming and the dependency on BCL2 in both Molm14 and MV4-11 cell lines (Figure 1A).To investigate the global transcriptomic changes induced by allosteric SHP2 inhibition, we performed total mRNA sequencing on Molm14, MV4-11 and SKNO1 cell lines. GSEA analysis revealed that RMC-4550 significantly upregulated expression of genes repressed by RAS activation, downregulated MYC targets, but also dysregulated genes mediating apoptosis. The most consistently upregulated pro-apoptotic gene was BMF (fold change: 4.39, FDR<0.001). BMF is a BH3-only protein found to be sequestered to motor filaments that, in response to cellular damage signals, is translocated in the cytoplasm and binds pro-survival Bcl2 proteins. The BMF transcript upregulation was confirmed by qPCR and western blot analysis showed a marked overexpression of the BMF protein level upon SHP2 inhibition, particularly in the cytoplasmic subcellular compartment (Figure 1B).We next treated Molm14, MV4-11, Kasumi and SKNO1 lines with incremental doses of RMC-4550 and Venetoclax in an 8x8 combination matrix to assess the synergy of the two compounds using cell viability and apoptosis readouts. The assay showed highly synergistic activity in both FLT3-ITD and KIT lines. Remarkably, we noted a potent synergy in Molm14 cells with concurrent mutation in NRAS G12C (Figure 1C).In a Molm14 cell line xenograft model, we demonstrated that the combination of RMC-4550 (30 mg/kg) and Venetoclax (100 mg/kg) administered orally 5 times a week for 28 days significantly decreased leukemia burden and improved survival (p<0.001) compared to control and single agents (Figure 1D).In a FLT3-ITD AML patient-derived xenograft (PDX) model, the combination of RMC-4550 and Venetoclax markedly decreased %hCD45 in both cardiac blood and spleen of NSGS mice compared to vehicle-treated control (Figure 1E).Supporting a potential therapeutic index for the combination, RMC-4550 and Venetoclax strongly inhibited colony formation in FLT3 AML primary samples compared to samples from healthy volunteers.Collectively, our data suggest that SHP2 inhibition increases the apoptotic dependency on BCL2 through up-regulation of the pro-apoptotic BMF, a mechanistic rationale to synergistically inhibit both targets. We provide preclinical evidence that co-targeting SHP2 and BCL2 is a potential effective therapeutic strategy in RTK-driven AML. [Display omitted] DisclosuresStahlhut: Revolutions Medicine: Current Employment, Current equity holder in publicly-traded company. Smith: Daiichi Sankyo: Consultancy; Amgen: Honoraria; AbbVie: Research Funding; Revolutions Medicine: Research Funding; FUJIFILM: Research Funding; Astellas Pharma: Consultancy, Research Funding.

Abstract NG06: Addressing scaffolding roles of oncogenic kinases with targeted protein degradation

Abstract Proteolysis Targeting Chimera (PROTAC) technology has emerged over the last two decades as a powerful tool for targeted degradation of proteins. PROTACs are heterobifunctional small molecules which co-opt the ubiquitin proteasome system to destroy oncogenic proteins. The development of PROTACs for receptor tyrosine kinases (EGFR, Her2 and c-MET), a protein family previously thought to be intractable for induced protein degradation, will be described. The use of VHL-recruiting PROTACs against this protein family reveals several advantages of degradation over inhibition alone including more potent inhibition of cell proliferation, a more durable and sustained downstream signaling response. Combined, these findings demonstrate the ability to target receptor tyrosine kinases for degradation using the PROTAC technology and outline the advantages of this degradation-based approach. Application of the PROTAC technology to targets, including RTKs, in hematological malignancies has also provided potential therapeutic approaches and enhanced understanding of scaffolding roles of oncogenic kinases. The receptor tyrosine kinase FLT-3 is frequently mutated in acute myeloid leukemia; however, traditional small molecule inhibitors suffer from limited efficacy in the clinic. A FLT-3 targeting PROTAC with sub-nanomolar antiproliferative activity and the ability to induce tumor regression in vivo will be described. Additionally, the discovery of apoptosis suppression as a kinase independent activity of FLT-3 ITD will be described. While the use of kinase inhibitors of oncoprotein BCR-Abl have enabled durable responses in patients with chronic myeloid leukemia (CML), issues of drug resistance and residual leukemic stem cells remain. An allosteric BCR-Abl PROTAC demonstrates significant advantages compared to inhibition and identified scaffolding roles for BCR-Abl which are not addressed by inhibitors but can be abrogated by protein degradation. Concurrent treated with the allosteric PROTAC and orthosteric inhibitors proved synergistic, particularly against some clinically relevant BCR-Abl kinase domain mutants. Furthermore, investigation into the kinase independent roles of BCR-Abl in patient stem cells suggest that these persistent leukemic stem cells are BCR-Abl agnostic. Citation Format: George Burslem. Addressing scaffolding roles of oncogenic kinases with targeted protein degradation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr NG06.

Abstract 2933: Inhibition of FLT3 and AXL by gilteritinib controls systemic tumor growth in luciferase-transduced MV-4-11 acute myeloid leukemia

Abstract Mutations and amplification of the fms-like receptor tyrosine kinase FLT3 gene, such as those found in MV-4-11 cells, are common in acute myeloid leukemia (AML). Such mutations result in aberrant activation of FLT3, increased growth and survival of tumor cells, and the development of refractory forms of AML with poor patient outcomes. In addition, AXL receptor tyrosine kinase imparts chemotherapeutic resistance in AML. MV-4-11 is a human biphenotypic B myelomonocytic leukemia with t(4;11)(q21;q23) chromosomal translocation commonly found in AML patients; it possesses a homozygous in-frame tandem repeat insertion in the FLT3 allele resulting in constitutively phosphorylated FLT3. Commonly employed preclinical orthotopic models of AML are primarily evaluated based on morbidity and mortality, which provide limited real time information on tumor burden and dissemination. The purpose of this study is to develop a MV-4-11 mouse model that allows the quantification and anatomical localization of the AML cells by monitoring bioluminescent signal in live animals and to utilize this model to investigate the efficacy of a FLT3/AXL inhibitor, gilteritinib. This poster describes a) the production of luciferase-expressing human MV-4-11 (MV-4-11-luc) cells via lentiviral transduction, b) the characterization of MV-4-11-luc progression in two models of immunocompromised mice, c) the efficacy of the FLT3/AXL kinase inhibitor gilteritinib against systemic MV-4-11-luc compared with two standard AML therapies, cytarabine and cyclophosphamide and d) the development of a humanized MV-4-11 tumor bearing model in NCG mice. Bioluminescence imaging revealed dose dependent tumor growth localized to the hind limbs and peritoneal cavity, which disseminated and intensified over time. Characteristic moribundity associated with disease progression was observed in this model. By comparison, robust growth of both parental and luc-transduced MV-4-11 was also observed in the highly immunodeficient NCG mice with or without prior myeloablation. Efficacy studies in NCG mice bearing parental or luciferase-tagged MV-4-11 cells demonstrated potent anti-tumor activity using the FLT3/AXL inhibitor. Bioluminescent imaging of orthotopic MV-4-11-luc xenografts in immunocompromised mice is a quantitative and sensitive method that provides anatomical context for assessing the efficacy of AML therapeutics. Ongoing and future work is focused on a) genetic profiling of MV-4-11 tumors after treatment with gilteritinib, b) further characterization of a humanized MV-4-11 model to evaluate immune-modulating therapeutics, and c) utilizing the model to evaluate activity of cell therapies that target AML. Citation Format: Na Li, Vivek Mahajan, Lindsay Butler, Elizabeth Rainbolt, Shasta Kidder, Kay Meshaw, Diana Gietl, Susan Yeyeodu, Anya Avrutskaya, Jing Wan, Thi Bui, Chassidy Hall, David P. Harris. Inhibition of FLT3 and AXL by gilteritinib controls systemic tumor growth in luciferase-transduced MV-4-11 acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2933.

A Unique N-Phenylbenzamide Inhibitor Targeting Non-Receptor Tyrosine Kinases Potently Suppresses AML Cell Growth In Vitro and In Vivo

Acute myelogenous leukemia (AML) is a hematologic cancer correlated with advancing age and poor prognosis. While diverse genetic changes are associated with AML, activating mutations in the Flt3 receptor tyrosine kinase are associated with nearly one-third of cases and include internal tandem duplication (ITD) of the regulatory juxtamembrane region. Additionally, recent studies have implicated several non-receptor tyrosine kinases in AML oncogenic signaling, including Fes, Syk and the myeloid Src-family members Fgr, Hck and Lyn. While selective Flt3 kinase inhibitors have been developed for clinical use, they have not produced durable responses, highlighting the need for new therapeutic approaches. In this study, we investigated the therapeutic potential of a multi-targeted tyrosine kinase inhibitor, TL02-59, which is based on an N-phenylbenzamide scaffold. Kinome-wide target specificity profiling (KINOMEscan) demonstrated a relatively narrow overall specificity profile for TL02-59, with only 23 potential targets out of 456 kinases when tested at a concentration of 1 µM. To confirm the results of the kinome-wide profiling against AML-associated kinases, we tested TL02-59 using in vitro kinase assays with recombinant Fes, Syk, Fgr, Hck, and Lyn as well as wild-type and ITD forms of the Flt3 kinase domain. TL02-59 displayed remarkable potency against the myeloid Src-family kinases Fgr and Lyn, with IC50values in the 100 pM range. TL02-59 also inhibited Fes, Syk, Hck and both forms of Flt3 in vitro, albeit with IC50 values in the 150-400 nM range. We then tested TL02-59 for growth-suppressive activity against Flt3-ITD+ AML cell lines, MV4-11 and MOLM-14, and the Flt3 wild-type cell line THP-1. Remarkably, TL02-59 inhibited the proliferation of MV4-11 cells with an IC50 value of 780 pM, with slightly lower potency against the MOLM-14 cells (IC50 = 3.5 nM). Quantitative real-time RT-PCR (qPCR) and immunoblot analysis confirmed that the AML-associated TL02-59 target kinases identified in vitro, including Fgr and Lyn, are expressed and active in MV4-11 and MOLM-14 cells. In contrast, TL02-59 had no effect on THP-1 cell growth up to 1 µM, consistent with transformation of these cells by kinase-independent mechanisms. We next examined the growth suppressive activity of TL02-59 in 26 bone marrow samples from AML patients, half of which expressed Flt3-ITD and half of which expressed wild-type Flt3. Twenty of the primary AML samples responded to TL02-59 treatment with growth inhibitory IC50 values in the nM range. We then determined the relative expression levels of all possible TL02-59 target kinases from the KINOMEscan analysis in these primary AML samples by qPCR. We discovered a striking correlation between TL02-59 sensitivity and expression levels of Fgr, Hck, and Lyn, supporting the idea that these Src-family members are the primary targets for TL02-59 in AML. In contrast, no correlation was observed with Flt3 expression, consistent with the observation that the four most sensitive patient samples were wild-type for Flt3. To test the efficacy of TL02-59 in vivo , we used immunocompromised (NSG) mice engrafted with MV4-11 AML cells as a model system. Two weeks after MV4-11 cell engraftment, mice were treated once daily with TL02-59 at 1 or 10 mg/kg by oral gavage for a total of 21 days. The mice were then sacrificed and evaluated for MV4-11 cells in their peripheral blood, spleen, and bone marrow by flow cytometry and IHC. At 1 mg/kg, TL02-59 reduced MV4-11 engraftment in the bone marrow by 20% and spleen engraftment by half, while suppressing the number of leukemia cells in the peripheral blood by 70% compared to vehicle control animals. At 10 mg/kg, TL02-59 reduced bone marrow AML cells by 70% and completely eliminated leukemic cells from the spleen and peripheral blood. Overall, these results demonstrate that TL02-59 is an effective inhibitor of AML cell growth in vitro and in vivo with a mechanism of action independent of Flt3 mutational status. The primary kinase targets for this inhibitor appear to be myeloid Src-family kinases, including Fgr, Lyn and Hck. DisclosuresNo relevant conflicts of interest to declare.

FLT3 Mutation in Acute Myeloid Leukemia: Primary or Secondary Mutation?

The receptor tyrosine kinase FLT3 gene is commonly mutated in acute myeloid leukemia (AML) and its mutation is associated with significantly more aggressive disease and poor outcome. However, in the co-presence of NPM1 mutation, the disease is less aggressive and is associated with better outcome. FLT3 inhibitor midostaurin (Rydapt; Novartis Pharmaceuticals, Inc.) was recently approved for patients with FLT3 mutations. While the presence ofa FLT3 mutation is commonly associated with AML,it can develop in less acute myeloid neoplasms, such as MDS and CMML, and implies transformation into more acute phase. In this context, the FLT3 mutation is typically detected in a subclone of the original chronic myeloid clone. The prevalence of subclonal FLT3 mutations is unknown, as is the clinical relevance of subclonal FLT3, especially when treated with FLT3 inhibitors. We used variant allele frequency (VAF) and next generation sequencing (NGS) to determine if FLT3 mutations are founding or secondary mutations and to characterize the co-mutational background in large number of consecutive patients who presented with a clinical impression of myeloid neoplasm.Methods: A total of 6390 consecutive bone marrow aspirate samples or peripheral blood samples were submitted with clinical impression of AML, MDS, or MPN between January 2014 and mid 2017 were tested for mutations in myeloid genes using NGS. We used the TruSight Myeloid panel (Illumina, San Diego, CA) for detecting missense mutations and fragment length analysis (FLA) for detecting internal tandem repeat (ITD) in FLT3 . DNA was extracted from samples using the QIAamp DNA Mini Kit. This NGS testing covers mutations in 54 myeloid-related genes. The average depth of sequencing was 10,000x.Results: A total of 2670 (42%) of the samples showed a mutation in one or more of the tested genes. Of the samples with mutations, FLT3 ITD and kinase domain (KD) mutations were detected in 313 (12%) patients. Of these patients, 208 (66.5%) had ITD and 117 (37.4%) had KD mutations. Twelve patients (3.8%) had both ITD and KD mutations. NPM1 mutation was detected in 138 (44%) of the patients. Most of FLT3 -KD mutations were in codon 835, but mutations in codons 836, 839, 842 and 845 were also found. Additionally, missense mutations in codons 576, 592, and 593 at the site of ITD were detected. To distinguish a FLT3 mutation as a founding mutation rather than a secondary mutation, we used a lower VAF of 10% as a cut-off. Using this approach, we found that 27% (115/313) of patients had secondary FLT3 mutations. Of these subclonal FLT3 mutants, NPM1 co-mutation was detected in 61 (53%) patients, but was not statistically significant (P=0.06). Patients with FLT3 mutation as a subclone had a significantly higher number of co-mutated genes (P-value <0.0001). This group of patients had higher (P≤0.01) mutations in TET2, DNMT3A, NRAS, IDH2, EZH2, and BCOR, consistent with an MDS/CMML background. On the other hand, patients with both FLT3 and NPM1 mutations had a significantly lower number of co-mutant genes (P<0.0001) and particularly lower (P≤0.01) mutation rate in U2AF1, SF3B1, SRSF2, RUNX1, IDH1, BCOR, and AXL1 .Conclusion: FLT3 mutations are frequently (27%) a secondary mutation in a subclone of the neoplastic founding mutations. This appears to be more common when FLT3 mutation is developing in a background of MDS/CMML. The co-presence of an NPM1 mutation is more commonly associated with fewer co-mutations in MDS-related genes. This data suggests that in addition to detecting ITD by FLA, using NGS for profiling mutations in FLT3 -KD and other myeloid genes should be considered whenever FLT3 mutations are evaluated. Further, the clinical decision for combination therapy should consider the entire mutation profile and not only mutations in FLT3 . [Display omitted] DisclosuresMa:NeoGenomics: Employment. De Dios:NeoGenomics: Employment. Funari:NeoGenomics: Employment. Sudarsanam:NeoGenomics: Employment. Jiang:NeoGenomics: Employment. Agersborg:NeoGenomics: Employment. Hummel:NeoGenomics: Employment. Blocker:NeoGenomics: Employment. Albitar:NeoGenomics: Employment.